Loss of Guanylyl Cyclase C (GCC) Signaling Leads to Dysfunctional Intestinal Barrier

BackgroundGuanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.
Methodology/Principal FindingsParacellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC−/−) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN−/−) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC−/− and UGN−/− mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC−/− mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC−/− and UGN−/− mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.
Conclusions/SignificanceGCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.

背景 经由尿鸟苷素（UGN）与鸟苷素激活的鸟苷酸环化酶C（GCC）信号通路，是调控肠液稳态、肠细胞增殖/凋亡及肿瘤发生的关键介导通路。针对上述部分效应的作用机制，我们提出假说：GCC信号通路可介导肠屏障功能的调控。
方法与主要结果 我们分别对经脂多糖（LPS）刺激或未刺激的野生型（WT）、GCC基因敲除（GCC−/−）小鼠，以及UGN基因敲除（UGN−/−）小鼠的肠段细胞旁通透性进行了检测。通过实时定量PCR（real-time PCR）测定了干扰素γ（IFNγ）与肌球蛋白轻链激酶（MLCK）的表达水平。在肠上皮细胞（IECs）及肠黏膜中，我们检测了紧密连接蛋白（TJPs）的表达、肌球蛋白II调节轻链（MLC）的磷酸化水平，以及信号转导与转录激活因子1（STAT1）的激活情况。在Transwell小室滤膜上培养的Caco-2与HT-29肠上皮细胞单层，我们分别在存在或不存在GCC RNA干扰（RNAi）的条件下检测了其通透性。结果显示，与野生型小鼠相比，GCC−/−及UGN−/−小鼠的肠通透性显著升高，同时伴随肠上皮细胞中IFNγ水平、MLCK及STAT1激活程度的上升。脂多糖刺激可使GCC−/−小鼠肠上皮细胞的IFNγ及STAT1激活水平较野生型小鼠进一步升高。GCC基因敲除小鼠的肠组织中，Claudin-2与JAM-A的表达量降低；且与野生型小鼠相比，GCC−/−及UGN−/−小鼠肠上皮细胞的磷酸化MLC水平显著升高。GCC基因沉默可诱导肠上皮细胞单层的MLC磷酸化，在基础条件下升高细胞单层通透性，并增强肿瘤坏死因子α（TNFα）与IFNγ诱导的细胞单层高通透性。
结论与意义 GCC信号通路通过调控MLCK的激活及紧密连接解离，在肠黏膜屏障完整性中发挥保护作用。因此，激活GCC信号通路或可成为维持小肠屏障以应对损伤的全新潜在机制。